Genetic instability is a hallmark of cancer. We have shown in previous work that the hypoxic tumor microenvironment is one cause of this genetic instability, and we have recently found that hypoxia specifically causes decreased expression of the DMArepair factors, RAD51 and BRCA1. These factors maintain genomic integrity by mediating the repair of double-strand breaks and other lesions through the high-fidelity homology-dependent repair (HDR) pathway. Germline mutations in BRCA1 have been linked to hereditary breast and ovarian cancer, and decreased expression of BRCA1 has been seen in many sporadic cancers of these sites. In this project, we propose to investigate the mechanisms and consequences of RAD51 and BRCA1 regulation in response to hypoxia. We will examine key transcription regulatory mechanisms, with a focus on promoter repression by E2Fs and associated pocket proteins, and we will test the hypothesis that RAD51 and BRCA1 are coordinately regulated by these factors. The impact of decreased RAD51 and BRCA1 expression on genetic instability will be assessed using selected DNA recombination and repair assays to test the hypothesis that hypoxia-induced decreases in RAD51 and/or BRCA1 may shift the balance between the high-fidelity HDR and the error-prone non-homologous end-joining repair pathways. We will also test whether the suppression of HDR in hypoxic cells renders them especially vulnerable to novel therapeutic strategies, including inhibition of base excision repair (BER) (in collaboration with Project 3) by either poly(ADP-ribose) polymerase-1 (PARP-1) or polymerase beta inhibitors (to cause accumulation of unrepaired strand breaks) or inhibition of O6-alkylguanine-DNA alkyltransferase (AGT) in combination with agents that target the O-6 position of guanine and give rise to crosslinks (in collaboration with Project 1). These studies will provide further insight into how the hypoxic tumor microenvironment contributes to genetic instability and may offer an explanation for the down-regulation of BRCA1 detected in sporadic cancers. This work may also serve as a basis for novel therapeutic strategies to target hypoxic cells in solid tumors and possibly to treat familial breast and ovarian cancers associated with mutations in BRCA1 or BRCA2.